A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell only-syndrome

Abstract Background The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), referring to lack of all spermatogenic cells in testes. Genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. Methods Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into germ cell-like cells. Results The homozygous loss-of function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking of all functional domains, and no PIWIL2 expression was detected in the patient’s testes. The patient and PIWIL2−/− hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses proposed inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2−/− group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. Conclusions Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.